Nuclear pore complexes (NPCs) are large, intricate structures that allow regulated exchange of macromolecules between the nucleoplasm and cytoplasm. Each complex, estimated to be 44 MDa in budding yeast, consists of approximately 30 proteins in multiple copies. Throughout the cell cycle, these complexes must continue to assemble and insert in the double lipid bilayer that surrounds the nucleus. This proposal seeks to elucidate the mechanism by which NPCs are assembled. In the first aim, global regulators of NPC biogenesis will be identified using yeast genetic approaches. A series of assays will be performed to determine the function of the proteins in assembly. The second aim focuses on establishing an In vitro model system for NPC assembly, which makes use of recently obtained yeast lysates containing vesicles believed to be NPC precursors. In the third aim, this in vitro assay will be applied to determine discreet stages required for yeast NPC assembly, and to identify roles of various proteins hypothesized to play a role in assembly. The proposed experiments utilize state of the art genetic approaches coupled with biochemistry, constituting a powerful approach to identifying key mediators of NPC biogenesis.